Our previous investigations of the molecular mechanisms of dysregulated lung cell apoptosis in emphysema led us to identify a marked increase in sphingolipid ceramide species in the lungs of patients with emphysema and in cells exposed to cigarette smoke (CS). Our findings implicated ceramide as an amplifier of oxidative stress and apoptosis in the lung. We postulate that ceramide is an important proximal mediator of CS-induced oxidative stress and injury in lung alveolar cells and therefore a putative therapeutic target in emphysema. Therefore, understanding the mechanisms by which CS upregulates ceramides in the lung is of utmost importance. We hypothesize that CS stimulates ceramide synthesis by dysregulating lipid interactions within the cellular membranes. Furthermore, we hypothesize that ceramide is a mediator of early alveolar lung injury induced by cigarette smoke characterized by alterations of the alveolar barrier function. We will investigate these hypotheses with the following specific aims: Specific Aim 1. To determine the biophysical mechanism by which CS upregulates ceramides, we will conduct biophysical investigations in membrane lipid bilayer models complemented with cellular injury models;and Specific Aim 2. To establish that ceramides are mediators of the CS-induced injury of the alveolar cell barrier function, we will study the capacitance of primary alveolar cell monolayers in real-time and we will develop and utilize for the first time an intravital application of two-photon emission microscopy (TPEM) of the intact rat lung. When completed, this work will elucidate the connection between bio-mechanical changes in the lipid bilayer membrane and biochemical events leading to ceramide signaling and, by implementing intravital TPEM imaging of the lung for the first time, it will accelerate biomedical lung research by its broad application to multiple mechanisms of lung health and disease. PUBLIC HEALTH RELEVANCE: We will investigate how cigarette smoke triggers the increase in ceramides in lipid membranes which are lining the cells and important organelles within the cells and are the sites of ceramides synthesis. Besides using classical methods, we propose a novel technique of real time recording of barrier function in the intact lung utilizing an advanced imaging modality to study if the increase in ceramide is a necessary step in the earliest lung injury induced by cigarette smoke. Our work is expected to provide the rationale for a therapeutic strategy that targets ceramide in patients with COPD and will develop a new intravital molecular imaging modality of the whole lung which can be applied to the studies of all lung diseases.